Device for separating plasma or serum from whole blood and analyzing the same

ABSTRACT

A device for separating plasma or serum from whole blood and analyzing the same, wherein the device includes a layer of glass fibers with an average diameter of from 0.2 to 5 microns and having a density of from 0.1 to 0.5 g./cm 3  and an adsorption volume at least twice that of the plasma or serum to be separated and, preferably, more than 3.3 times the volume.

This application is a continuation of application Ser. No. 608,991,filed May 10, 1984, now abandoned, which in turn is a divisional ofapplication Ser. No. 289,943, filed Aug. 4, 1981 and now U.S. Pat. No.4,477,575, which was issued Oct. 16, 1984.

BACKGROUND OF THE INVENTION

This invention relates to a device for separating plasma or serum fromwhole blood.

In clinical chemistry, the separation of serum or plasma from wholeblood is of outstanding importance since, in practice, it is notpossible to carry out the analysis of dissolved blood components withoutdisturbances unless the separation takes place.

The normal and most conventional manner of separating serum or plasmafrom erythrocytes is centrifuging. Centrifugation, however, causesproblems with respect to separating supernatant and blood cake. Thisproblem is particularly acute when small amounts are involved. To thatend, a whole series of adjuvants have been described in the literature.See, in this regard, Federal Republic of Germany Patent SpecificationNo. 25 59 242 (U.S. Pat. No. 4,012,325).

The use of whole blood in the case of rapid diagnostic agents gives riseto special problems. Rapid diagnostic agents are reagent-containingcarriers, which are absorbent or swellable. Preferably, they are made offilter paper. In use, one applies a small amount, such as a droplet, ofthe liquid to be investigated to the carrier. After a short period oftime to allow reactions to take place, a color change occurs, which canbe evaluated visually or via remission photometry. Since turbid orcoloured solutions, such as blood, disturb the readings, attempts have,therefore, been made to make rapid diagnostics available for the directuse of whole blood. Thus, for example, mention may be made of thecoating of test papers with semi-permeable membranes (see U.S. Pat. No.3,092,465) and the use of swellable films into which only the dissolvedcomponents of the blood can penetrate but not the erythrocytes (seeFederal Republic of Germany Patent Specification No. 15 98 153; U.S.Pat. No. 3,630,957). These two methods are per se usable but only fortests for low molecular weight components of blood, for example glucoseor urea. Higher molecular weight components of the blood, such aslipids, or substrates bound to serum protein, such as bilirubin, cannotbe determined in this way because they are not able to penetrate intothe film or to pass through the semipermeable membrane. Furthermore,suggestions have been made for covering diagnostic agents with membranefilters for separating off the blood cells (see Federal Republic ofGermany Patent Specifications Nos. 22 22 951 and 29 22 958 whichcorrespond to U.S. Pat. Nos. 3,663,374 and 4,256,693). A disadvantage ofthese diagnostic agents is that the blood can only penetrate through themembrane filter very slowly and in small amounts because the membrane isvery easily blocked up. This results in a reaction which takes longerthan is desired. In contradistinction to the previously-mentioneddiagnostic agents, which are already commercially available, "rapidtests" of the last-mentioned type have not yet been marketed.

Federal Republic of Germany Patent Specification Nos. 29 08 721 and 2908 722 which correspond to U.S. Pat. Nos. 4,246,107 and 4,330,410, teachthat lymphocytes and leukocytes can be separated from blood when bloodis filtered through a layer of synthetic resin fibres with an averagefibre diameter of 5 to 20μ (lymphocytes) and of 3 to 10μ (leukocytes).However, since the erythrocytes preponderantly pass through the filterwith the plasma, these filters are not suitable for obtaining plasma.Furthermore, carbon fibres, glass fibres and metal fibres are mentionedpurely speculatively.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a simplemeans for separating plasma or serum from whole blood which separatessmall amounts of blood quickly and without centrifuging and isespecially suitable for preparing samples for diagnostic purposes.

It has now been found that the separation of plasma or serum from wholeblood takes place quickly and simply and in sufficient amount when theblood is allowed to run through a packing of glass fibres alone or inadmixture with other fibres. This must be regarded as being surprisingsince, in the above-mentioned Federal Republic of Germany PatentSpecification No. 22 22 951, the use of glass fibre mats has alreadybeen described for the separation of white corpuscles but, in order toseparate erythrocytes, this reference teaches that a membrane filter isabsolutely necessary.

Thus, this invention teaches a device useful in separating plasma orserum from whole blood, comprising a layer of glass fibres with adiameter of 0.2 to 5μ, preferably of 0.5 to 2.5μ, and especially of 0.5to 1.5μ and with a density of 0.1 to 0.5 g./cm³, the volume of the lasmaor serum to be separated being at most 50% and preferably less than 30%of the absorption volume of the glass fibre layer; i.e., the absorptionvolume of the glass fibre layer is at least two times and preferablymore than 3.3 times the volume of the plasma or serum separated from thewhole blood.

The glass fibres can be in loosely stapled form, or can also be in theform of papers, fleece or felts or can also be formed in any desiredshape when held by a forming body.

Rapid diagnostic devices which use the thus described formed glassfibres can be used with whole blood. Previously, it was necessary toobtain serum or plasma from whole blood in order to use a rapiddiagnostic agent. This is now unnecessary.

Furthermore, columns, filter funnels or other suitable vessels packedwith glass fibres can also be used for obtaining serum or plasma bysimply allowing whole blood to run through. This makes serum or plasmaavailable for diagnostic agents since the serum or plasma passes throughsuch a layer more quickly than the erythrocytes and leukocytes.

The above-mentioned glass fibres can consist of fibres of differingdiameters. The glass material can consist of alkali-containing oralkaline-free boro-silicate glass or pure quartz glass. Fibre of othertechnical glasses, for example boron-free alkali glasses, crystal glass,lead glass and the like, are not commercially available with thenecessary dimensions and could not be investigated. However, it isassumed that they are also suitable. The average diameter of the glassfibres can be from 0.2μ to about 5μ, preferably from 0.5μ to 2.5μ andespecially from 0.5μ to 1.5μ. The diameters of the fibres can, dependingupon their mode of production, vary very considerably but an upper limitof about 10μ should only be exceeded in exceptional circumstances. Thelength of the fibres is only limited by the nature of the packing.Depending upon the nature of the packing, densities of 0.1 to 0.5 andusually of 0.2 to 0.4 g./cm³ are found.

Furthermore, the glass fibres can be mixed with one another and alsowith other materials, the internal holding together of the fibresthereby being improved. Thus, for example, use can be made of syntheticfibres, such as of polyester, polyamide and the like, as well as fibresof polyethylene, polypropylene and other thermoplastic synthetic resins.These additional fibres can also have a larger diameter (e.g. 10 to20μ), so long as the amount thereof is not so large that it adverselyaffects the seprating ability of the finer glass fibres used accordingto the present invention.

Furthermore, the glass fibres can be consolidated by the addition ofinorganic binding agents, for example waterglass, or of organic bindingagents, for example polyvinyl acetate, polyvinyl propionate, polyacrylicacid esters and the like, by means of which the glass fibres are stuckat the points of contact to the binder.

The combination of the glass fibre layers according to the prsentinvention with diagnostic agents is especially preferred and is also thesubject of the present invention.

In these diagnostic agents, the glass fibres can also contain reagentswhich prevent the haemolysis of erythrocytes, as well as reagents whichinhibit or promote coagulation. The glass fibres can also containreagents which are required in the indicator layer but are incompatiblewith other reagents present therein. These latter reagents can, ofcourse, also be placed in layers which are between the glass fibres andthe indicator layer.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention will now be described withreference to drawings, in which:

FIG. 1 is a schematic elevation of a first preferred embodiment;

FIG. 2 is an elevation of a second preferred embodiment;

FIG. 3 is an elevation of a third preferred embodiment;

FIG. 4 is an elevation of a fourth preferred embodiment;

FIG. 5 is a top view of the fourth preferred embodiment shown in FIG. 4;

FIG. 6 is an elevation of a fifth preferred embodiment;

FIG. 7 is a top view of the preferred embodiment shown in FIG. 6;

FIG. 8 is an elevation of a sixth preferred embodiment;

FIG. 9 is an elevation of a seventh preferred embodiment;

FIG. 10 is an elevation of an eighth preferred embodiment;

FIG. 11 is an elevation of a ninth preferred embodiment;

FIG. 12 is an elevation of a tenth preferred embodiment;

FIG. 13 is an elevation of an eleventh preferred embodiment;

FIG. 14 is an elevation, partly in section, of a twelfth preferredembodiment;

FIG. 15 is an elevation, partly in section, of a thirteenth preferredembodiment;

FIG. 16 is an elevation, partly in section, of a fourteenth preferredembodiment;

FIG. 17 is a top view of the fourteenth preferred embodiment shown inFIG. 16;

FIG. 18 is an elevation, partly in section, of a fifteenth preferredembodiment;

FIG. 19 is an elevation, partly in section, of a sixteenth preferredembodiment;

FIG. 20 is an elevation, partly in section, of a seventeenth preferredembodiment;

FIG. 21 is an elevation, partly in section, of an eighteenth preferredembodiment;

FIG. 22 is an elevation, partly in section, of a nineteeth preferredembodiment;

FIG. 23 is an elevation, partly in section, of a twentieth preferredembodiment;

FIG. 24 is an elevation, partly in section, of a twenty-first preferredembodiment.

FIG. 25 is a top view of the embodiment shown in FIG. 24;

FIG. 26 is an elevation, partly in section, of a twenty-second preferredembodiment;

FIG. 27 is a top view of the embodiment shown in FIG. 26;

FIG. 28 is an elevation, partly in section, of a twenty-third preferredembodiment;

FIG. 29 is a top view of a twenty-fourth preferred embodiment;

FIG. 30 is an elevation, partly in section, of a twenty-fifth preferredembodiment; and

FIG. 31 is an elevation partly in section, of a twenty-sixth preferredembodiment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The construction of one such diagnostic agents according to the presentinvention is illustrated in FIG. 1 of the accompanying drawings. Onto astiff substrate 2, the reaction layer 1 is stuck. Closely above thereaction layer, a thin and inherently stable separating layer 4 isapplied which is permeable to liquids. This stable separating layerconsists of a meshwork of woven or felted synthetic resin fibres, isstuck on both sides of the reaction layer, and is positioned on thesubstrate in an easily removable manner and in such a way that an easilygripped end 4a on the longer end of the substrate remains free. Abovethe reaction and separating layers, a glass fibre paper 3 is applied.This is fixed in position by a further meshwork 5 which, like theseparating layer 4, is stuck down on either side of the reaction layer.

The reaction layer 1 can consist of an impregnated absorbent carrier orof a swellable or porous synthetic resin film. The substrate 2 ispreferably a comparatively thick synthetic resin film, a rigidcardboard, a glass plate or some other stable carrier material.

After the application of a drop of blood 8 to the upper side of therapid diagnostic agent, the plasma is separated from erythrocytes andleukocytes in the glass fibre paper 3. The plasma separated in thismanner passes via the separating layer 4 into the reaction zone or layer1 of the diagnostic agent. After an appropriate time, during which theplasma has penetrated into the reaction zone, the free end 4a of theseparating layer is gripped and the layer 4 removed, together with theglass fibre paper 3 and the meshwork 5. Subsequently, the reaction layerin which the reaction now takes place can be evaluated visually orremission photometrically.

Another possible form of construction of the rapid diagnostic agentaccording to the present invention is illustrated in FIGS. 2 and 3. Inthis embodiment, in addition to the above-described construction, theyhave one or more layers 6 which are permeable to all kinds of liquids.These layers are applied above (FIG. 2) or below (FIG. 3) the glassfibre paper 3. These additional layers can be impregnated with reagentswhich are either readily soluble and, together with the plasma, passinto the reaction zone or are less soluble and allow one or morepreliminary stages of the detection reaction to take place outside ofthe final reaction zone 1.

FIGS. 4 and 5 illustrate another construction of the rapid diagnosticagent according to the present invention, in which the glass fibre paper3 is stuck directly on to the substrate 2. The reaction layer 1 issuperposed on one zone of the glass fibre paper defined thereby. Blood 8is applied to the other zone of the glass fibre paper which remainsfree. The plasma separating from the erythrocytes in the glass fibrepaper diffuses in the glass fibre paper towards the reaction layer andpasses into it. The colours resulting due to the reaction can beobserved from the upper side of the rapid diagnostic agent and can beevaluated. The reaction layer can be applied directly to the glass fibremat by "printing" or coating. However, it can also be stuck on to theglass fibre paper in the form of a wholly or partly impregnatedabsorbent carrier.

Furthermore, as illustrated in FIGS. 9 and 10 of the accompanyingdrawings, the diagnostic agent according to the present invention can beconstructed in such a manner that onto the substrate 2, an absorbentmaterial 9 is first applied, such as cellulose paper or a syntheticfibre fleece. Above this material, the glass fibre paper 3 and thereaction layer 1 are applied. The absorbent material 9 can have the samesurface area as the reaction layer (FIG. 10) or can have a largersurface area so that the material 9 has an uncovered area (FIG. 9). Theblood 8 is droped on to the uncovered surface area of the absorbentmaterial (FIG. 9) or directly next to the absorbent material (FIG. 10)and rapidly taken up by this and sucked under the glass fibre paper.Subsequently, due to the absorbency of the glass fibre paper, blood issucked upward through the glass fibre paper, separation of theerythrocytes thereby taking place, and plasma passes into the reactionlayer 1. The reaction is, as in FIG. 4, observed from the upper side ofthe rapid diagnostic agent.

FIG. 11 of the accompanying drawings illustrates yet anotherconstruction of a rapid diagnostic agent suitable for the direct use ofwhole blood. This figure shows an embodiment in which an absorbent layer9 and glass fibre layer 3 are applied onto a stiff substrate 2, side byside. The absorbent layer may consist of e.g., cellulose paper or acellulose containing synthetic fibre fleece. The two layers should be inclose contact with one another. On the surface of the absorbent layer 9,reaction layer 1 the detection contains reagents needed for rapiddiagnosis. These reagents can be applied, for example, by coating withan open film as described in Federal Republic of Germany PatentSpecification No. 29 10 134. Upon applying a drop of blood 8 on to theside of the glass fibre layer remote from the reaction zone, theseparation of plasma from erythrocytes takes place in such a manner thatthe plasma front first reaches the point of separation from theabsorbent layer 9 and is immediately sucked up by this. Due to capillaryforces, the plasma then passes into the actual reaction layer 1 wherethe detection reaction becomes visible, for example, by a colour change,which can be seen from above.

Further, FIG. 14 depicts another embodiment of the present invention.The glass fibre paper 3 is stuck onto a substrate 2, the substratehaving one or more holes 11 at the point of contact. On the other sideof the glass fibre paper, a reaction layer 1 is applied either directlyor by sticking. Blood 8 is applied to the diagnostic agent in such amanner so that it can pass through the hole or holes to the glass fibrepaper 3. The plasma separated in the glass fibre paper now passes to thereaction layer 1 and gives rise to a reaction which can be evaluated onthe surface thereof visually or remission photometrically. The reactionlayer can also be protected by a transparent covering layer 7 as shownin FIG. 15.

The reaction layer 1 can be, for example, a layer "printed" on to theglass fibre mat and can also be a multi-layer element in which thelayers can contain various reagents and/or can fulfil several functions,as is described, for example, in Federal Republic of Germany PatentSpecification No. 29 22 958 (U.S. Pat. No. 4,256,693).

FIG. 16 of the accompanying drawings shows a side view and FIG. 17 a topview of a further construction of a diagnostic agent according to thepresent invention. In this embodiment the layer 3 consists of glassfibres. Additionally, one or more layers 6 necessary for the reactioncan be held together by a previously produced formed body 12. The blood8 is dropped on to the glass fibre filter side of the diagnostic agent.The separated plasma subsequently passes into the reaction layer 1 andthe indicator reaction on the side opposite to the glass fibre filtercan be evaluated visually or remission photometrically.

For reasons of clarity of the drawings, in some Figures (for exampleFIGS. 1 and 16) the various layers are, in part, shown with intermediatespaces. In practice, however, the layers lie on one another in such amanner that liquids can pass unhindered from one layer to another.Furthermore, in FIG. 16, the reaction layers 1 and 6 are illustrated asdivided horizontally in order to indicate that they can also consist ofseveral layers lying on top of one another.

Blood separation accomplished using the agents according to FIGS. 4, 5,9 and 11 can be substantially improved when a hydrophobic barrier isapplied on the glass fibres 3 beside or in the reaction layer 1 whichbarrier 15 partly projects into the glass fibres 3. FIGS. 6, 7, 12 and13 show these barriers 15 but otherwise correspond in their constructionto that of FIGS. 4, and 11. This barrier not only prevents blood 8 onthe surface of the glass fibres from spreading out and contaminating theindicator zone, but it also decisively improves the separating action ofthe glass fibres. Therefore, it is possible to operate with relativelysmall glass fibre papers 3, the result of which is that substantiallysmaller amounts of blood are required. The barrier can be applied in aconventional manner, for example from nozzles or with rollers. Thehydrophobic material for the barrier can be, for example, a meltadhesive, a conventional solvent adhesive or wax.

Furthermore, FIG. 18 of the accompanying drawings illustrates theconstruction of a formed body according to the present invention whichseparates plasma from the erythrocytes of whole blood withoutcentrifuging and makes the plasma available for diagnostic purposes. Forthis purpose, a tube or a vessel 13, which has, for example, theillustrated shape, is packed with the glass fibres 14. Blood 8 isintroduced into the vessel from above. On the downward path, the plasmais separated from the erythrocytes of the blood by means of the glassfibres. The plasma collecting in the lower part of the vessel 13 cannow, for example, be withdrawn or sucked off through an "end-to-end"capillary 13a and used directly for diagnostic purposes.

Another construction according to the present invention is illustratedin FIG. 19 of the accompanying drawings. In this embodiment vessel 13appropriate for the separation of the erythrocytes has the form of apiston syringe, the lower part of which is tightly packed with glassfibres 14. The blood 8 is introduced into the open top of the vessel.After separation of plasma and erythrocytes has taken place, the plasmawhich collects in the lower part of the vessel can be forced out of thesyringe into capillary 13a' by the insertion careful pressing of apiston 17.

The process according to the present invention for obtaining plasma, asillustrated in FIG. 20 of the accompanying can also be carried out withthe use of a vessel 13". It is divided into two parts by a valve 16which operates to permit passage in one direction, the vessel 13" beingpacked with the above-described glass fibres 14. The blood 8 isintroduced into the top of the vessel. After separation from theerythrocytes, the plasma collects in the lower part of the vessel andcan then be removed by pressing together the lower part of the vessel,the valve 16 preventing the plasma from flowing back into the upper partof the vessel containing the blood cells.

Furthermore, the process according to the present invention can becarried out with an arrangement as illustrated in FIG. 1, the reactionlayer 1 stuck on to the substrate 2 thereby consisting of a definiteabsorbent material so that, upon applying blood, a definite volume ofplasma passes into the layer 1. After separation of the layers 3, 4 and5, the plasma, i.e. the substance to be analyzed, can be eluted by asolvent. The elution of the plasma and the analysis can be carried outimmediately but also, depending upon the substance to be analyzed, atsome later time in another place. If the analysis is to take placelater, it can be advantageous first to dry in the plasma, for examplewith warm air or by freeze drying. Furthermore, it is possible,separately to provide one or more zones separate from the zone forsample application which contains reagents so that, when eluting with asolvent, the whole reaction mixture is simultaneously eluted.

If the reaction colour is to be evaluated not only visually but also ina remission photometer, it is preferable to cover the reaction layer 1with a covering layer 7 in order to avoid contamination of themeasurement arrangement. If the reaction layer 1 is a film, for exampleas described in Federal Republic of Germany Patent Specifications Nos.29 10 134 or 15 98 153, then it is preferable to coat this directly onto the covering layer 7 e.g. FIGS. 8, 15, and 21) and then assemble thetwo together to the substrate. One possible embodiment is illustrated inFIG. 8 of the accompanying drawings. Of course, the covering layer 7 canalso be used in other embodiments, such as is illustrated in FIG. 15which otherwise corresponds to the construction of FIG. 14.

It has proven to be advantageous to choose arrangements in which thereaction layer 1 is not brought into fluid-conveying contact with theglass fibre layer 3 of the absorbent layer 9 until said layers 3, 9 arecompletely filled with plasma or serum. The advantages of thesearrangements are that the plasma or serum can be brought into contactwith the reaction layer 1 at a previously determined and exact time.Furthermore, this contacting takes place over the whole surface area sothat chromatographic effects, such as can occur in the case of the abovearrangements, are prevented. The fact that, between the application ofthe blood 8 and the commencement of the reaction in the reaction layer1, there can be a previously determined time is of great importance inthe case of reactions which are to take place under specially controlledconditions. Thus, in the case of the determination of enzymes, whichmust take place at especially constant temperature, the reaction canfirst be commenced when the diagnostic agent has a sufficiently constanttemperature. Furthermore, the layers 3 and 9 in which the plasmacollects can be provided with reagents which bring the substances to bedetected into a particular state by a time-dependent reaction, i.e.permit a preliminary reaction to take place to completion. One exampleof this is the activation of creatine kinase with N-acetylcysteine.

FIGS. 21, 22 and 23 of the accompanying drawings illustrate variouspossible embodiments of the invention. FIG. 22, the hydrophobic barrier15 simultaneously serves to secure layers 1 and 7. The description andcomposition of the other layers correspond to FIGS. 4 to 13.

FIG. 23 illustrates the use of a hydrophobic mesh 18 between thereaction layer 1 and glass fibres 3 or the absorbent layer 9. Thishydrophobic mesh protects the arrangement against unintentional slightcontact and only permits liquid contact to take place upon applyingpressure. This results in an improved practicability.

Of course, it is possible that the reaction layer 1 consists of two ormore different zones (e.g. FIGS. 1 to 3 and 16). These can containeither the same substance in different concentration ranges or differentsubstances when the formulations are appropriately selected.Furthermore, it is also possible to make constructions in which variousreaction layers are simultaneously moistened by the plasma emanatingfrom a single point of application (e.g. FIG. 24 et seq.). In this case,a large variety of shapes can be used, such as longitudinal, circular orthe like.

FIGS. 24 to 29 of the accompanying drawings illustrate some of thesepossible arrangements, the various test zones being indicated by 1a to1d (appropriately primed), the other components corresponding to thoseof the previously described Figures.

Blood separation can also be improved when, a further glass fibre zone3a is applied as in FIGS. 30 and 31 at that point where blood is droppedthereupon. In this case, layers 3a and 3 can consist of the samematerial but for 3a a material can be chosen with a different thicknessor with a different fibre diameter.

FIGS. 30 and 31 of the accompanying drawings illustrate possiblearrangements which otherwise generally correspond to FIGS. 8 and 21.

The following Examples are given for the purpose of illustrating thepresent invention.

EXAMPLE 1 Cholesterol test strips

0.117 g. methyl hydroxyproylcellulose (Culminal HMPC 8000)

7.000 g. titanium dioxide

0.138 g. monopotassium dihydrogen phosphate

0.479 g. disodium monohydrogen phosphate hydrate

3400 U cholesterol esterase

5000 U cholesterol oxidase

7×10⁴ U peroxidase

0.476 g. sodium dioctyl sulphosuccinate

are dissolved in 70 ml. water. There are then successively homogeneouslyincorporated

14.0 g. cellulose

8.4 g. polyvinyl propionate dispersion.

Finally, there is added

0.66 g. 3,3',5,5'-tetramethylbenzidine, dissolved in 1.6 ml. acetone.

This batch is coated in an approximately 300μ thick layer on to a smoothsynthetic resin film and, after drying at 60°-70° C., cut up into 6 mm.wide strips. Subsequently, these strips, together with a 60μ thickmeshwork of nylon and a glass fibre paper (glass fibre filter No. 3362of Schleicher & Schull; paper thickness 0.47 mm., density 0.27 g./cm³,average fibre diameter about 2.5μ), also cut up into 6 mm. wide strips,are stuck on to a polyester film. Subsequently, it is cut up into 6 mm.wide strips.

If 40 μl. of blood are applied to the upper side of the test strip and,after 1 minute, the glass fibre with the residual blood is removed,together with the meshwork, by tearing off, then, within 3 minutes, areaction colour forms on the test zone which corresponds to that whichis obtained when, instead of the blood, use is made of plasmacentrifuged off from the same blood.

EXAMPLE 2 Cholesterol test

0.45 g. monopotassium dihydrogen phosphate

1.55 g. disodium monohydrogen phosphate hydrate

1.5×10⁴ U cholesterol esterase

1×10⁴ U cholesterol oxidase

3×10⁵ U peroxidase

2.0 g. sodium dioctyl sulphosuccinate

6.9 g. sodium alginate (Algipon)

are dissolved in 250 ml. water and then 2.0 g.3,3',5,5'-tetramethylbenzidine, dissolved in 15 ml. acetone, are addedthereto, whereafter 20.0 g. kieselguhr are homogeneously distributedtherein. This reaction mass is applied in 6 mm. wide strips with a silkscreen printing machine (fabric: 190μ) on to a glass fibre paper (forexample glass fibre filter No. 85/90 of Machery, Nagel & Co.) in themanner described in Example 1. The "printed" glass fibre paper is driedat 60° to 80° C. and then cut up into 12 mm. wide strips in such amanner that the "printed" reaction zone accounts for one half of thestrip. This strip is stuck on to the end of the polyester film and thisis then cut up into 6 mm. wide strips at right-angles to the glass fibrepapers. When 40 μl. of blood are now dropped on to the edge of theuncoated glass fibre paper remote from the reagent layer, the plasmadiffuses under the reaction zone. Depending upon the cholesterolconcentration of the blood, this reaction zone now assumes a bluereaction colour of differing strong colour. The intensity of thereaction colour corresponds to that which is obtained when, instead ofthe blood, one uses a serum or plasma obtained from the same blood.

The papers described in the following Table 1 can be used in the sameway:

                  TABLE 1                                                         ______________________________________                                        Glass fibre papers for plasma separation                                      paper                                                                                         fibre   average                                                                              wt. per                                                        dia-    fibre  surface                                                                             thick-                                                                              density                            manu-           meter   diameter                                                                             area  ness  (g/                                facturer                                                                              type    (μ)  (μ) (g/m.sup.2)                                                                         (μm)                                                                             cm.sup.3)                          ______________________________________                                        Machery &                                                                             85/90   2-9     3.2    87    350   0.249                              Nagel   BF                                                                    Nuclepore                                                                             P300    1-3     1.5    25     89   0.275                              Schleicher                                                                            No. 9   3-4     3.4    67    269   0.247                              & Schull                                                                      Schleicher                                                                            3362    1-7     2.5    127   472   0.269                              & Schull                                                                      ______________________________________                                    

EXAMPLE 3 Cholesterol test

A reagent mass consisting of

16.0 g. cellulose M+N Ac 10

86.0 g. of a 0.2% solution of methylhydroxypropyl cellulose (CulminalMHCP 8000)

0.32 g. wetting agent (Marlon)

0.68 g. wetting agent (sodium dioctylsulphosuccinate)

12.0 g. polyvinyl propionate dispersion (Propiofan 70 D)

0.48 g. 3,3',5,5'-tetramethylbenzidine

10.0 g. titanium dioxide

9600 U cholesterol esterase

7200 U cholesterol oxidase

1.04×10⁴ U peroxidase

0.01 g. gallic acid

is coated in a thickness of 0.2 mm. on to a hydrophobic polyester fleece(Reemay 2033, du Pont) and dried at 60° C. Subsequently, a 6 mm. widestrip of this coating and a 12 mm. wide strip of glass fibre filter(e.g. Filter 3362 of Schleicher & Schull) are stuck side by side on afirm plastics strip in such a manner that the glass fibre filter is veryclose to the coated fleece. When strips of 6 mm. width are cuttransversely from this plastics strip, test strips are obtained withwhich, after dropping about 50 μl. whole blood onto the side of theglass fibre filter remote from the reagent fleece, after a short timeonly pure plasma passes over to the reagent fleece and results in theformation of a blue reaction colour, the intensity of which increaseswith the concentration of the cholesterol in the blood.

EXAMPLE 4 Separate recovery of plasma

A synthetic resin vessel which downwardly narrows conically (e.g. asynthetic resin tip with a piston pipette, length 5 cm., thickness 0.5cm.) is loosely filled two thirds full with glass fibres according tothe following Table 2, packing densities of 0.1 to 0.4 g./cm³ beingobtained. After the free, upper part has been filled with blood, theserum diffuses into the tip of the vessel. From there, an "end-to-end"capillary of 15 μl. capacity can be filled by attachment to the openingof the pipette tip. The plasma obtained in this manner can now be useddirectly for any desired analytical process.

                                      TABLE 2                                     __________________________________________________________________________    Investigation of the separating ability of various glass fibres in an         experimental arrangement according to Example 4.                              Glass fibres: properties, separating abilities for erthrocytes/plasma         Johns-                                                                             VEB diameter (μm)                                                                        fibre                                                                             Surface                                                Manville                                                                           Trisola  average                                                                            length                                                                            area separation                                        USA  DDR range*                                                                             value                                                                              μm                                                                             m.sup.2 /g                                                                         erythrocytes/plasma                               __________________________________________________________________________    100       0.2-0.29                                                                          0.25  300                                                                              5.1  +                                                 102       0.3-0.33                                                                          0.32     4.5  +                                                 104      0.34-0.48                                                                          0.4   800                                                                              3.12 +                                                 106      0.49-0.58                                                                          0.54 1000                                                                              2.6  +++                                                    U 60                                                                              0.51-0.64                                                                          0.58     --   ++                                                108A     0.59-0.88                                                                          0.74 1200                                                                              1.72 ++                                                     U 70                                                                              0.65-0.76                                                                          0.71          ++                                                     U 80                                                                              0.77-0.93                                                                          0.85          +++                                                    U 90                                                                              0.94-1.19                                                                          1.07          ++                                                     U 100                                                                              1.2-1.44                                                                          1.32          +++                                               108B     0.89-2.16                                                                          1.53     0.71 +++                                                    U 156                                                                             1.45-2.49                                                                          1.97     --   +                                                 110      2.17-3.10                                                                          2.6      0.49 -                                                 112      2.6-3.8                                                                            3.2  1900                                                                              0.40 -                                                      F   2.5-4.0                                                                            3.3           -                                                 __________________________________________________________________________     + satisfactory;                                                               ++ good;                                                                      +++very good;                                                                 - negative.                                                                   *80% of the fibres lie within this range                                 

EXAMPLE 5 Effects of a hydrophobic barrier

To demonstrate the effects of a hydrophobic barrier 15, an arrangementaccording to FIG. 14 was assembled comprising a transparentpolycarbonate film 2 of 6 mm. width and 0.3 mm. thickness, a 9×6 mm.absorbent layer 9 of 0.09 mm. thick glass fibre paper, a hydrophobicnylon meshwork 18 of 0.075 mm. thickness and a transparent covering film7. A glass fibre paper 3 is fixed in absorbent contact on to thecarrier, which paper 3 consists of a glass fibre paper (Schleicher &Schull, No. 3362, thickness 0.47 mm.) of 6 mm. width and with the lengthgiven in the following Table 3. In each case, one half of these devicesare provided on the point of contact between the layers 3, 9 and 18 withan approximately 2 mm. wide and 0.1 mm. thick barrier 15 of paraffinwax.

For the determination of the amount of plasma obtained, on the middle ofthe glass fibre paper 3 there are applied the amounts of blood indicatedin the following Table 3 and, after 30 seconds, the wetting of theabsorbent layer 9 is determined, as well as any possible supersaturation(depth of penetration of the erythrocytes into layer 9). The resultsgiven in Table 3 show that, due to the hydrophobic barrier, theseparation is improved and a complete saturation is achieved so thatsuch a test can be carried out even in the case of very small volumes ofblood, for example with capillary blood from a finger tip. In each case,the experiments were carried out five times and the results determined.

                                      TABLE 3                                     __________________________________________________________________________                 % wetting of layer 9 with                                                                  % wetting of layer 9 without                        dimensions of                                                                        volume of                                                                           hydrophobic barrier                                                                        hydrophobic barrier                                 the glass fibre                                                                      blood (μl)                                                                       plasma blood plasma blood                                        fleece (3)                                                                           applied                                                                             .sup.--X n = 5                                                                       .sup.--X n = 5                                                                      .sup.--X n = 5                                                                       .sup.--X n = 5                               __________________________________________________________________________    6 × 6 mm.                                                                      25    88     0     77     12                                                  30    97     0     72     22                                                  35    99     0     83     18                                           7 × 6 mm.                                                                      29    86     0     82      3                                                  35    100    0     87     21                                                  41    100    0     97     31                                           8 × 6 mm.                                                                      33    96     0     79      0                                                  40    100    0     92      0                                                  47    100    0     100    13                                           __________________________________________________________________________

It will be understood that the specification and examples areillustrative but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

What is claimed is:
 1. A unitary multilayer device for separating plasmaor serum from whole blood and analyzing such plasma or serum,comprising:(a) a fibrous layer capable of separating blood cells fromserum or plasma in a whole blood sample, wherein said fibrous layercontains glass fibers and has a density of from 0.1 to 0.5 g/cm³ andwherein said glass fibres have an average diameter of from 0.2 to 5microns; and (b) a reaction layer which is or may be brought into fluidcommunication with said fibrous layer while leaving at least a portionof said fibrous layer free from direct attachment to said reactionlayer, said reaction layer contaning means for determining a componentof plasma or serum.
 2. The device of claim 1, further comprising aninert substrate to which said fibrous layer and said reaction layer areattached directly or indirectly.
 3. The device of claim 1, wherein saidglass fibers have an average diameter of from 0.5 to 2.5 microns.
 4. Thedevice of claim 1, wherein said glass fibers have an average diameter offrom 0.5 to 1.5 microns.
 5. The device of claim 1, wherein said fibrouslayer further comprises synthetic resin fibers.
 6. The device of claim1, wherein said reactin layer contains a plurality of layers.
 7. Thedevice of claim 1, further comprising an inert carrier between saidfibrous layer and said reaction layer which carrier permits passage ofplasma or serum into said reaction layer.
 8. The device of claim 1,further comprising an inert carrier layer positioned below said reactionlayer wherein said reaction layer and said inert carrier are adapted toproduce a detection reaction with a component material of plasma orserum which is analyzable or detectable through said inert carrier. 9.Device of claim 1, further comprising a removable layer which supportssaid glass fiber containing fibrous layer.
 10. The device of claim 9,further comprising means for fixing said removable layer to a substrate.